KR20100106744A - A composition for preventing or treating migratory skin cell or melanin synthesis related diseases - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of diseases related to mobile skin cells or melanin synthesis.

Description

Composition for preventing or treating migratory skin cell or melanin synthesis related diseases}

The present invention relates to a composition for the prevention or treatment of diseases related to mobile skin cells or melanin synthesis.

The skin is largely composed of keratinocytes cells, which actually make up the skin, and melanocytes (melanocytes) that produce melanocytes in relation to skin color. In contrast to the identity of keratinocytes, melanocytes are representative mobile skin cells, and abnormal increase or decrease in their mobility causes various forms of disease, collectively referred to as mobile skin disease.

The research on mobile skin cell disease is much more active in Korea than in Korea because of much more serious social needs than in Korea. In fact, in 2002 in Europe, about 60,000 new melanoma cases were diagnosed and 17,000 died. In 2004, nearly 60,000 new cases were diagnosed in the United States and nearly 7,000 were affected by the disease. It is expected to die. In the United States, NIH's NIAMS (www.niams.nih.gov.) Has a framework to support research into the causes, treatment, and prevention of overall skin diseases, including vitiligo (vitiligo), Scott of the Cold Spring Harbor Institute. Lowe and his team have targeted the Apaf-1 study, which induces apoptosis of malignant melanoma cells.The University of Pittsburgh Cancer Research Institute has been treating the patients with malignant melanoma by immunomodulators histamine dihydrochloride and interleukin-2. I'm working on IDM Pharmaceuticals has begun Phase II clinical trials for patients with stage 2 and 3 melanoma who have undergone a resection surgery for melanoma vaccines, which are being jointly developed with Aventis in 2005. Shering-Plough is hepatitis C. Intron A (interferon alpha-2b), which is approved in Europe as a therapeutic agent, is being developed as a therapeutic agent for melanoma. However, the level of technology for research and development of therapeutics in terms of cell mobility control, which is a fundamental cause of mobile skin disease, is still poor.

The mobility of all cells depends on their migration potential and extracellular matrix remodeling ability, which is signaled by adhesion of the cell adhesion receptor and extracellular matrix on the cell surface. It is tightly controlled. This failure to regulate cell mobility in vivo is expressed in a variety of diseases, for example, the decrease in cell mobility of melanocytes by changes in extracellular matrix with aging causes cell aging and pigmentation, while melanin Abnormal decrease in cell mobility causes vitiligo and abnormal increase in melanocytes causes malignant melanoma.

Vitiligo is an acquired depigmentation disease in which white spots of various sizes and shapes appear on the skin due to a lack of melanocytes (Le Poole IC, Boissy RE, Sarangarajan R, Chen J, Forristal JJ, Sheth P, Westerhof W, Babcock G, Das PK, Saelinger CB.In Vitro Cell Dev Biol Anim. 2000; 36 (5): 309-19; Yu HS.J Biomed Sci. 2002; 9 (6): 564-73.), The most common of skin diseases. Representative disease. Vitiligo is present in about 1% of the population (Whitton ME, Ashcroft DM, Barrett CW, Gonzalez U. Cochrane Database Syst Rev. 2006; 1: CD003263), and 400,000 people in Korea suffer from vitiligo. Nevertheless, vitiligo has not yet been found to be a satisfactory therapeutic effect and has been recognized as an extremely refractory disease.

Melanocytes, the main cause of vitiligo, are cells in the lower part of the epidermis. One is every 10 keratinocytes. Under normal conditions, melanocytes play a role in making and delivering pigments to keratinocytes. Deficiency causes depigmentation in various tissues including skin.

The cause of vitiligo is still unknown, but the cause of the disease has been explained to date. It is known that the immune system is caused by autoimmunity and the excess of the chemical mediators that are released near melanocytes. There is a humoral theory of melanocytes, and melanocyte autologous destruction of melanin-forming intermediates or metabolites that accumulate in melanocytes and destroy melanocytes. (Sanlı H, Akay BN, Arat M, KoP, Akan H, Beksac M, Ilhan O. Dermatology. 2008; 216 (4): 349-354; Machado Filho CD, Almeida FA, Proto RS, Landman G. Vitiligo: Sao Paulo Med J. 2005; 123 (4): 187-91; Schallreuter KU, Bahadoran P, Picardo M, Slominski A, Elassiuty YE, Kemp EH, Giachino C, Liu JB, Luiten RM, Lambe T, Le Poole IC, Dammak I, Onay H, Zmijewski MA, Dell'Anna ML, Zeegers MP, Cornall RJ, Paus R, Ortonne JP, Westerhof W. Exp Dermatol. 2008; 17 (2): 139-40). There is also a genetic predisposition that is more easily damaged.

However, this theory is thought to cause vitiligo by inducing melanocyte deficiency by acting in combination rather than independently.

Currently widely used treatment for vitiligo is ultraviolet light treatment (Rivard J, Hexsel C, Owen M, Strickland FM, Lim HW, Hamzavi I. Photo adaptation of vitiliginous skin to targeted ultraviolet B phototherapy.Photodermatol Photoimmunol Photomed. 2007; 23 (6): 258-60), epidermal grafts that remove blisters from the normal site and then implant into the vitiligo site. Photochemistry, which involves applying drugs, ointments, creams, and then UV light, requires long-term treatment, whereas epidermal transplantation has the advantage of being able to finish the treatment in a short time, but it is expensive.

Photochemistry, which is currently widely used, is a treatment method that irradiates UV rays after taking medicines such as methoxsalen and psoralen, which are photosensitizers that make skin sensitive. At this time, ultraviolet rays may also regenerate melanin pigment in some cases. In the case of UV treatment, there is a 50-70% therapeutic effect on the face, torso and upper limbs, while the limbs are less effective and usually require long-term treatment for more than a year, usually twice a week.

Over the years, vitiligo has been studied in biochemical, immunological, genetic, and various biological aspects. Nevertheless, the exact mechanism of expression has not been elucidated yet, and phototherapy using ultraviolet rays is widely used, but it does not have a full therapeutic effect. Although the phototherapy by ultraviolet irradiation resulted in a measurable increase in the number of melanocytes, this was only a limited and partial increase. Moreover, even if the melanocyte count increased, the proliferated melanocytes were transferred to the lesion site of the alum. There is little effect of vitiligo treatment by substantially no migration.

Therefore, in order to effectively treat vitiligo, there is a need for a method capable of promoting the proliferation of acquired melanocytes that have been destroyed, and at the same time, inducing proliferation of melanocytes proliferated to the lesion site with vitiligo.

To do so, it is necessary to establish a basis for controlling the mobility of melanocytes by identifying new regulators that regulate melanocyte mobility through research on melanocyte migration regulation mechanisms. For therapeutic applications, it is considered the easiest way to control the identified mobility modulators and to screen readily applicable natural substances. If this process is successfully performed, it is thought that the treatment of incomplete vitiligo by UV irradiation can be greatly improved.

Another consideration in the treatment of vitiligo is the regulation of melanin synthesis. Because melanin content synthesized by melanocytes ultimately determines the skin color. In fact, melanin synthesis is applied to a variety of skin whitening.

Interest in skin whitening has been continued since the Egyptian, Greek, and Roman eras, and can be easily found in Korean history. Skin whitening, especially in conjunction with well-being, has increased social attention, and half of Asians aged 25-34 are spending billions of dollars on whitening cosmetics.

Harmful potential from lead, mercury, hydroxyquinone and steroid hormones, which are currently widely used and used in whitening agents, has been raised, and the boundaries of direct bleaching side effects are being raised (Ly F, Soko AS, Dione DA, Niang SO, Kane A, Bocoum TI, Dieng MT, Ndiaye B. Int J Dermatol. 2007 46 Suppl 1: 15-7.Edith Nnoruka and Obiefuna Okoye.J Natl Med Assoc. 2006 98 (6): 934939. Cheng SL, Huang Liu R, Sheu JN, Chen ST, Sinchaikul S, Tsay G J. Biol Pharm Bull. 2006 29 (4): 655-69). Therefore, there is an urgent need to develop a safe whitening agent without toxicity.

In general, since skin color is controlled by melanin synthesized in melanocytes, non-cytotoxic and selective control of melanin synthesis is the most efficient and fundamental whitening agent development strategy.

Therefore, the present inventors have investigated the mechanism of regulation of cell mobility of melanocytes by syndecan-2 (syndecan-2), a cell binding receptor present on the cell surface, and the ability of melanocytes to be regulated through the regulation of synthecan-2 expression. By promoting the development of cell mobility modulators for the treatment of mobile skin diseases such as vitiligo.

In addition, the mechanism of regulating melanin synthesis by syndecane-2 and the regulation of syndecan-2 expression regulators will be presented to suggest the possibility of developing a whitening agent using the regulation of melanin synthesis in the future.

The present invention has been made in view of the above problems, and the object of the present invention is to provide a composition for treating or preventing mobile skin disease.

Still another object of the present invention is to provide a composition for skin whitening.

In order to achieve the above object, the present invention provides a composition for the treatment or prophylaxis of mobile skin disease comprising a syndecane-2 protein consisting of SEQ ID NO: 1 as an active ingredient.

In an embodiment of the present invention, the mobile skin disease is preferably one or more diseases selected from the group consisting of melanoma, skin pigmentation, skin pigmentation and vitiligo, but is not limited thereto.

In another aspect, the present invention is synthesized in SEQ ID NO: 2 Provided are a composition for treating or preventing mobile skin diseases using a gene as an active ingredient.

Provided is a composition for the treatment or prevention of mobile skin diseases, comprising as an active ingredient a gene for at least one syndecane-2 selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 6.

In another aspect, the present invention provides a composition for skin whitening comprising an active ingredient syndecane-2 consisting of SEQ ID NO: 1.

In another aspect, the present invention provides a composition for skin whitening comprising a syndecane-2 gene consisting of SEQ ID NO: 2 as an active ingredient.

The present invention also provides a composition for skin whitening comprising the gene for at least one syndecane-2 selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 6 as an active ingredient.

In a preferred embodiment of the invention, the syndecan-2 protein is not only the amino acid sequence set forth in SEQ ID NO: 1 but also one or more substitutional, deletion, translocation, addition, etc. of the protein. A protein having an activity of 2 is also included in the scope of the protein of the present invention. Preferably, the sequence identity of the amino acid sequence disclosed in SEQ ID NO: 1 is 80% or more, 85% or more, 90% or more, 93% or more, 94% At least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

In the present invention, the polypeptide has a certain ratio (eg, 80%, 85%, 90%, 95%, or 99%) of sequence identity to another sequence, when aligning the two sequences, By comparison it is meant that the amino acid residues in this ratio are identical. The alignment and percent homology or identity may be determined by any suitable software program known in the art, such as those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al. (Eds) 1987 Supplement 30 section 7.7.18). Can be determined using Preferred programs include the GCG Pileup program, FASTA (Pearson et al. 1988 Proc. Natl Acad. Sci USA 85: 2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, MD, and Altschul et al. 1997 NAR25: 3389-3402. Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), which preferably uses basic parameters. Another sequence software program that can be used is the TFASTA Data Searching Program available from Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).

In addition, the present invention includes a gene encoding syndecan-2 having an amino acid sequence of SEQ ID NO: 1, and the gene sequence thereof is represented by SEQ ID NO: 2.

In addition to the gene encoding the mutant of Cindecan-2 having the amino acid sequence of SEQ ID NO: 1, as well as the mutant syndecan-encoding the mutant syndecan-2 protein obtained by mutating the amino acid sequence of SEQ ID NO: Two genes are also included in the gene which concerns on this invention.

In the case of the siRNA of the present invention, not only the genes described in SEQ ID NOs: 3 to 6, but also genes including all siRNAs affecting the activity of syndecane-2 produced by these mutations are included in the protection scope of the present invention. .

Hereinafter, the present invention will be described.

Cosmetics prepared with a cosmetic composition containing the peptide, gene or compound of the present invention as an active ingredient may be prepared in the form of a general emulsion formulation and solubilized formulation. Cosmetics of emulsion formulations include nutrient cosmetics, creams and essences.

Cosmetics of cosmetic formulations include soft cosmetics. In addition to cosmetics containing the peptides, genes or compounds of the present invention, by containing a dermatologically acceptable medium or base may be prepared in the form of adjuvants for topical or systemic application commonly used in the field of dermatology. .

Suitable cosmetic formulations include, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes), nonionics obtained by dispersing an oil phase in a solution, gel, solid or pasty anhydrous product, aqueous phase. It may be provided in the form of a vesicle dispersant, in the form of a cream, skin, lotion, powder, ointment, spray or conceal stick.

It may also be prepared in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition to the peptides, genes or compounds, the cosmetic composition of the present invention is in addition to fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, interfaces Active, water, ionic or nonionic

Cosmetics such as emulsifiers, fillers, metal ion sequestrants and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives, lipid vesicles or any other ingredients commonly used in cosmetics Or an adjuvant commonly used in the field of dermatology. And the above ingredients may be introduced in amounts generally used in the field of dermatology.

Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

The present invention also provides a melanogenesis inhibitor containing the composition as an active ingredient.

As a specific formulation of the melanogenesis inhibitor of the present invention, syrups, tablets, capsules, troches, liquids or suspensions may be used, but is not limited thereto.

In addition, the present invention provides a composition for skin whitening containing the composition as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for treating or preventing skin melanoma containing the composition as an active ingredient.

Pharmaceutical compositions of the present invention may be formulated for oral administration, for example, oral administration such as tablets, powders, capsules, troches, solutions, suspensions; Or it can be formulated into various preparations such as ointment. Pharmaceutical formulations prepared using conventional carriers can be administered orally or parenterally, for example, for dermal or topical application. In addition, the dosage of the active ingredient is different depending on the age, condition, etc. of the patient, but in general, 10 to 500, preferably 50 to 300 mg amount per day is administered to the adult, the judgment of the doctor or pharmacist Depending on the time interval can be administered several times a day, preferably once or six divided doses.

Peptides of the invention can be used in the preparation of dermatological and / or cosmetically active compositions. Such compositions comprise a peptide of the invention in the range of 0.5 ppm to 5,000 ppm (w / w), preferably 1 ppm to 1,000 ppm (w / w), based on the weight of the peptide and bulk agent of the invention. It is contained in an effective amount.

Peptides of the invention can be in the form of solutions, dispersions or emulsions; Or in a form encapsulated in a carrier such as macrocapsules, microcapsules or nanocapsules, liposomes or silomicrons; Macroparticles, microparticles or nanoparticles, or enclosed forms in microfungus; Or as adsorbed forms on powdered organic polymers, talc, bentonite, and other inorganic carriers.

Peptides of the present invention may be in any galenic form, e.g. emulsion, milk, lotion, ointment, viscous gelatinous lifting emulsifying polymer, pomade, shampoo, soap, gel, powder, stick It can be used in the form of sprays, body oils, face masks, or transdermal applications.

Peptides of the present invention may be used in other commonly used components, such as extracting lipids and / or synthetic lipids, viscous gelatinous lifting emulsifying polymers, water soluble or fat soluble actives, plant extracts, tissue extracts, marine extracts (marine) extracts, sun protection agents, antioxidants, humectants, and barrier agents, and skin-revitalizing actives.

Peptides of the present invention are used in all skin-related products for preventing the disease, and prevent the progress of the disease for drugs and / or cosmetics associated with vitiligo, skin whitening, skin cancer and the like.

When the peptides of the present invention are used as a cosmetic composition for skin whitening, the substance contains 0.001 to 5% (w / w), preferably 0.01 to 3% (w / w). It is difficult to expect a substantial whitening effect at concentrations less than 0.001%, and if it exceeds 5% concentration, it may adversely affect the stability of the formulation and the product due to low solubility.

Ethanol, Glycerine, Butylene Glycol, Propylene Glycol, Glyceres-26, Methylgluses-20, Isocetyl Myristate, Isocetyl Octanoate, Octyldodecyl as a solvent when preparing the composition for skin whitening of the present invention One or more selected from myristate, octyldodecanol, isostearyl isostearate, cetyloctanoate and neopentyl glycol dicaprate are used. When the composition of the present invention is prepared using such a solvent, the solubility of the compound in the solvent varies slightly depending on the kind of the compound and the mixing ratio of the solvent. The type and amount of usage can be selected and applied appropriately.

The composition according to the present invention may be prepared in the form of cosmetics such as skin external ointment, softening longevity, nourishing cosmetics, nutrition cream, massage cream, essence, pack. Here, the skin external ointment is prepared to contain 50.0 to 97.0% by weight of petrolatum and 0.1 to 5.0% by weight of polyoxyethylene oleyl-ether phosphate in addition to the active ingredient of the present invention, the softener is polyhydric alcohols such as propylene glycol, glycerin 1.0 To 10.0% by weight and 0.05 to 2.0% by weight of surfactants such as polyethyleneol rail ether and polyoxyethylene hydrogenated castor oil. Nutrients and nourishing cream contains 5.0 to 20.0% by weight of oils such as squalane, petrolatum and octyldodecanol and 3.0 to 15.0% by weight of wax components such as cetanol, stearyl alcohol and beeswax in addition to the active ingredients of the peptide of the present invention. The essence contains 5.0 to 30.0% by weight of polyhydric alcohols such as glycerin and propylene glycol. Massage cream is prepared by containing 30.0 to 70.0% by weight of oil such as liquid paraffin, petrolatum, isononylisononanoate, etc. in addition to the active ingredient of Formula 1 or 2, and the pack is 5.0 to 20.0% by weight of polyvinyl alcohol. It is prepared as a wash-off pack containing 5.0 to 30.0% by weight of pigments such as kaolin, talc, zinc oxide and titanium dioxide in the off-pack or general emulsified cosmetics.

The abbreviations used in the present invention have the following meanings: α-MSH, α-melanin cell stimulating hormone; FGF, fibroblast growth factor CD, conditioned media; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix FAK, focal adhesion kinase; FBS, fetal bovine serum; SFM, serum-free medium.

To elucidate the mechanism of melanocyte motility regulation and melanin synthesis regulation by syndecan-2 expression, ① primarily identify the mechanism of cellular mobility according to the change of syndecan-2 expression in melanoma or melanocytes, ② syndecan The mechanism of regulation of melanin synthesis in response to changes in -2 expression will be elucidated. ③ The melanin cell migration and melanin synthesis regulation by sindecan-2 will be examined in animal models.

Syndecane-2 is a cell binding receptor present on the surface of the cell, and the results of the present inventors confirmed that the expression of various cancers such as colorectal cancer, melanoma, sarcoma, etc. is increased to promote cell migration. The expression of syndecane-2 and the regulation of cell mobility in melanocytes are thought to have a significant correlation. First, overexpression of syndecane-2 in the melanoma cell line B16 cell line or reduction of syndecane-2 expression using syndecane-2 siRNA, followed by FACS and transwell migration assay. And thus, the cell mobility of melanoma cells was investigated. At this time, the expression of syndecane-2 was confirmed by FACS using syndecane-2 antibody, and cell mobility was analyzed by cell migration assay using transwell. The FACS and migration assay results were combined to investigate the correlation between syndecane-2 expression and cell migration of melanin and melanoma cells.

By performing the same experiment as the melanoma cells using primary melanocytes, it was verified whether the melanocyte mobility is regulated by syndecan-2 expression (see FIG. 11).

Melanocytes in skin tissue are regulated by keratinocytes. Therefore, cell mobility through transwells and melanocytes in the skin tissue where keratinocytes are present are unlikely to be the same. Therefore, it is intended to verify the mobility effect of melanocytes by natural products in artificial skin consisting of keratinocyte (Fig. 10).

Artificial skin is established by culturing a mixture of keratinocytes and melanocytes in a ratio on the collagen matrix, and the movement of melanocytes is confirmed through tissue sections and antibody staining (FIG. 10A). In addition, in order to investigate the effect of UVB used to treat vitiligo on melanocyte mobility, the effects of UVB were first identified by culturing keratinocytes and melanocytes pretreated with UVB (Fig. 10B), and then sequentially By using the selected natural products, the effect of the natural products by verifying the effect on the cell mobility of melanocytes through keratinocytes will be verified (Fig. 10C). Staining of melanocytes is selectively stained using the characteristics of the cells containing melanin.

We also revealed the function of syndecane-2 in melanoma. Overexpression of syndecane-2 increased the migration and invasion of melanoma cells, and the opposite effect was observed when synthecan-2 was knocked down using small inhibitory RNAs. Syndecane-2 expression was increased by fibroblasts growth factor-2, which is known to promote melanoma cell migration; α-Melanin cell stimulating hormone reduced independant-2 expression and melanoma cell migration and invasion independently of melanin-synthesis. Syndecane-2 overexpression also recovered the migration defect induced by α-MSH treatment. These data strongly suggest that syndecane-2 plays an important role in the mobility of melanoma cells.

A. Vitiligo and Cell Mobility:

Vitiligo is the most representative disease of acquired depigmented skin diseases in which various forms of white spots appear on the skin due to the lack of melanocytes, which occur in about 1% of the population. The treatment can be relatively improved by UV treatment using long-wave ultraviolet rays, but at least 100 long-term treatments should be performed at a frequency of 2-3 times a week, and surgical treatment such as skin grafts and inhalation blisters. In some cases, vitiligo is regarded as a refractory disease that does not expect satisfactory therapeutic effect. The most fundamental reason that treatment is not possible is that the peripheral melanocytes must be properly migrated after treatment because these migrations are not controlled. Therefore, the development of breakthrough technology to increase melanocyte mobility is essential for the treatment of vitiligo.

B. Malignant Melanoma and Cell Mobility:

Malignant melanoma, the most malignant of skin cancers, is a tumor in which melanin cells are malignant, and the number of annual occurrences is around 1000-1500 (1-1.5 per 100,000 population), and the number tends to increase year by year. Malignant melanoma, like other cancers, is important for early detection and early treatment. The most important means of treatment is surgical treatment by surgery, but malignant melanoma has very high cell mobility. As a result, tumors recur. Therefore, in order to treat malignant melanoma, it is necessary to first develop a technology for inhibiting their cell mobility.

C. Other Melanin Cell Mobility-Related Diseases:

Since the variation of cell mobility of melanocytes induces various forms of malignant melanoma and vitiligo in various tissues such as eyes, ears, and epidermis, the necessity of technology for controlling cell mobility of melanocytes is greatly increased.

D. Cell Mobility of Adsorbent Synthecan and Melanocyte Cells:

Syndecane is one of the cell adhesion receptors that regulate the binding of cells and extracellular matrix present on the surface of the cell. At the same time, as a component of the extracellular matrix, syndecan actively controls various physiological activities such as cell proliferation and migration differentiation. In particular, it is the only receptor that can directly control the cytoskeletal changes that are essential for the remodeling of extracellular matrix and cellular mobility. Through the study of the cell mobility control of cancer cells which the present inventors have performed for many years, syndecan-2, which is not expressed in normal epithelial cells with low cell mobility, increases expression in cancer cells with high cell mobility and regulates cell mobility. I confirmed the fact. In other words, the cell mobility of cancer cells is increased by receptor exchange, in which the receptor that inhibits cell mobility disappears and the expression of the receptor that increases cell mobility is increased during the cancerization process. Similar to this phenomenon, increased expression of syndecane-2 increased the cell mobility of melanocytes, and inhibition of the expression of syndecane-2 was found to decrease the cell mobility of melanocytes. This suggests that the regulation of medecan-2 expression alone can regulate the cell mobility of melanocytes.

In order to develop a therapeutic agent for treating pigmentation, vitiligo and malignant melanoma, which are representative skin diseases caused by abnormal changes in melanocyte cell mobility as a physiologically active protein application technology, (1) replacement of the cell adhesion receptor Based on the basic research that regulates cell mobility, the principles of effective cell mobility control are identified, and (2) effective melanocyte mobility is established by establishing technology to control the expression and cell mobility of cell binding receptors including syndecane. The purpose of this study is to develop a therapeutic agent for cell motility skin disease by searching for control substances and controlling melanocyte migration.

Abnormal variation of melanocyte mobility causes various skin diseases such as vitiligo, melanosis, leukoderma, and malignant melanoma in the skin, and also without control of melanocyte cell mobility Due to the fact that the treatment of the disease is impossible, the cell mobility control technology of the melanocytes is a key technology for the development of skin disease treatment. Cellular mobility control of melanocytes causes various forms of malignant melanoma, vitiligo, Harada's syndrome and Waardemburg syndrome in various tissues such as eyes, ears and epidermis. It can be applied to the development of therapeutic agents. In addition, since cell mobility abnormalities are the cause of asthma, arthritis, dementia, arteriosclerosis, cancer, etc., cell mobility control technology using the research results of the present subject matter has wide applicability for the development of therapeutics for various diseases. It has a technology base that can be applied to various fields of applied life science.

In general, various approaches are possible to develop a skin disease treatment agent. However, in the case of the mobile skin cell disease of the present invention, treatment cannot be performed without ultimately controlling cell mobility. This is the most essential and core technology.

Because the surface surface receptors have high substrate selectivity and function specificity and exist on the cell surface, they are widely recognized as the best targets for the development of selective new drugs because of the advantage of the extracellular point of action of the developed drug. Therefore, for the most efficient and selective cell mobility control, the development of cell adhesion receptor control technology is the core technology. In fact, bioactive receptor substitution technology with specific functions is a core technology of modern biotechnology, which is essential for regulating the function of genes and proteins involved in various diseases as well as skin diseases, and minimizes side effects. It is a key technology for developing skin disease treatments that can maximize efficiency.

Cell mobility is controlled by a combination of extracellular matrix remodeling and intracellular cytoskeleton. Syndecan is the only adhesion receptor that can directly control both. Therefore, cell mobility control by syndecane is the most efficient and key control technology.

We provide evidence for the tumorigenic activity of syndecane-2 using melanoma. The present inventors identified syndecan-2 expression in tissue samples derived from human melanoma but did not confirm its expression in normal melanocytes (FIG. 2A). Similarly, syndecane-2 expression was increased in all melanoma cell lines tested (FIG. 2B). Increased synthecan-2 expression correlates with increased melanoma cell migration but the opposite effect was observed when syndecan-2 expression was inhibited using siRNAs (Fig. 3 & 4). Thus, dedecane-2 is shown to act as a pro-tumorigenic adhesion receptor common in cancer cells.

Melanocytes and melanoma cells differ in cell surface expression of syndecane-2, a receptor that plays an important role in the migration of melanoma cells. Interestingly, syndecan-2 expression was regulated by α-MSH, a pituitary-derived peptide that promotes melanin synthesis. These results indicate that the reduction of syndecane-2 expression by α-MSH inhibits melanoma cell migration (FIG. 7). FGF-2 increased synthecan-2 expression, increasing melanoma cell migration (FIG. 6), while BPE or a-MSH inhibited syndecane-2 expression, reducing melanoma cell migration (FIG. 6). 6 & 7). Interestingly, α-MSH-mediated syndecane-2 expression occurs independently of melanin synthesis (FIG. 8). Α-MSH thus plays a unique role in the regulation of syndecane-2 expression in cell migration and melanin biosynthetic pathways.

Syndecan-2, a cell surface adhesion receptor, also plays an important role in regulating the migration capacity of melanoma cells. In addition, α-MSH inhibits the expression of syndecane-2 independently of the melanin biosynthetic pathway and simultaneously inhibits cell migration. These findings may provide clues to the tumor's ability to metastasize and contribute to the development of new drugs that reduce the metastasis of melanoma.

It was also confirmed that syndecane-2 is involved in the regulation of melanin synthesis. Cancerization by overexpressing syndecan-2 in normal skin cells (melanin cells) with relatively lower expression of syndecane-2 compared with malignant melanoma and malignant melanoma. And whether syndecane-2, which plays a role in cancer activity, induces melanin pigmentation in melanoma and melanocytes.

In addition, the observation of melanin synthesis change by syndecane-2 using tissue immunostaining was performed as follows; When syndecan-2 was overexpressed, their correlation was confirmed by immunostaining using syndecan-2 and melanosome marker (HBM-45) to observe the increased melanin in tissues.

As can be seen in the present invention, it can be seen that the cell surface adhesion receptor syndecan-2 plays an important role in the synthesis of mobile skin disease or melanin, thereby reducing the skin whitening, vitiligo and melanoma metastasis. Can contribute to the development of new drugs.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following Examples are described for the purpose of illustrating the present invention, the scope of the present invention is not limited by the following Examples.

Among the reagents and antibodies used in the present invention, monoclonal antibodies against vinculin, paxillin and phosphotyrosine (4G10) are described by Upstate Biotechnology, Inc. (Lake Placid, NY). MAb and phosphorylation site-specific FAK [PY ³⁹⁷ ] and polyclonal antibodies to FAK [PY ⁸⁶¹ ] for FAK are described in BioSource Quality Controlled Biochemicals, Inc. (Morgan Hill, Calif.). Monoclonal antibodies against human melanosomes (Clone HMB-45) were purchased from Dako (Denmark). b-galactosidase expressing plasmids (pCMV / β-gal) and Luciferase Reporter Gene Assay kits were prepared using Promega Corp. (Madison, WI), Effectene from Qiagen (Hilden, Germany), bovine pituitary extract (BPE) from Lonza (Visp, Switzerland), and α-MSH from Sigma Aldrich (St. Louis, MO). The anti-Ig HRP detection kit was purchased from BD Biosciences (San Jose, Calif.).

The production of monoclonal antibodies against the extracellular domain of syndecan-2-mAb against syndecan-2 was found in AdipoGen Inc. Produced by (Korea). Sites of murine syn-2 cDNA showing extracellular domain (S2E) were amplified by polymerase chain reaction and cloned upstream of human immunoglobulin heavy chain fixation site (Fc) was constructed in both pAGCF and pAGPCF2; The former used the native leader sequence and the latter used the plasminogen activator inhibitor-1 (PAI-1) leader peptide. Fc-fusion constructs were transfected with human embryonic kidney 293 (HEK293) cells. DMEM serum-free medium was used to collect murine sys-2-Fc fusion protein from confluent cells for 2 days. The medium was collected and spun to remove cells and the pH adjusted to 8.0 with 1M Tris. The medium was then filtered using 0.4-μm filter paper, loaded on a Protein-A column, eluted with 100 mM glycine, pH 3.0, and immediately neutralized with 1/10 volume of 1M Tris pH 8.0. Both constructs give a similar amount of rat syn-2-Fc. Balb / c mice were repeatedly immunized with S2E-Fc fusion protein until polyclonal serum showed a strong immune response from immunized mice. Splenocytes were isolated from mice and fused to mouse myeloma Sp2 / 0. Screening and single cell cloning followed established standard protocols. Ascites was obtained from immunocompromised Balb / c mice. Immunoglobulin fractions were obtained via Protein G. Three stable hybridoma clones (# 194, # 197 and # 302) secreting mAb against syndecane-2 were obtained (FIG. 1). Specificity and cross-reactivity for the mAbs were determined by Western blot analysis, slot blot analysis, FACS analysis and immunohistochemistry.

Example 1 Cell Culture and Transfection

Human colon adenocarcinoma cells HT-29 and CT116, human melanoma cells A375 and SK-MEL-28, mouse melanoma cells B16 and B16F10, and human fibrosarcoma HT1080 cells were purchased from the Korean Cell Line Bank. HT-29 cells were treated with McCoy's 5A complete medium (Life Technologies, Rockville, MD), 10% (v / v) fetal bovine serum (FBS), penicillin (100 units / ml) and streptomycin (10 μg / ml, Gibco BRL) At 37 ° C., under 5% CO ₂ conditions. RPMI (Life Technologies, Rockville, MD) given 10% (v / v) fetal calf serum (FBS) and gentamicin (50 μg / ml, Sigma) under A375, B16, B16F10, and 37 ° C., 5% CO ₂ conditions SK-MEL-28 cell line was maintained at and HT1080 cell lines were maintained at DMEM (Life Technologies, Rockville, MD). Transient transfection was performed using Effectene reagent (Qiagen) or LipofecATMINE 2000 (Invitrogen) according to the manufacturer's instructions.

Example 2 Stable Transfection of Syndecane-2 cDNA

HT29 cells were collected by trypsinization. The cells were washed twice with cold PBS and 3 × 10 ⁶ cells were incubated on ice for 20 min with a control- or syndecane-2 expression vector. Transfection was performed by electroporation using a period of 0.2 kV, 1 pulse time and 15 seconds. Clones were selected using G418 (1 mg / ml) for 3 weeks. Syndecan-2 expression was assessed by RT-PCR using syndecan-2 specific primers.

Example 3 Synthesis of Small Interfering RNA Constructs

Oligonucleotides targeting human syndecan-2 or mouse syndecane-2 comprising a 9-bp hairpin loop were designed. Oligonucleotides were annealed and cloned into pSUPER vectors. The sequences of the primers are: man-decane -2 new sense primer, 5'GATCCCC TGACGATGACTACGCTTCT TTCAAGAGA A CTGCTACTGATGCGAAGA TTTTTGGAAA- 3 '( SEQ ID NO: 3) Human-New decane -2 anti-sense primer, 5'AGCTTTTCCAAAAA TGACGATGACTACGCTTCT TCTCTTGAA ACTGCTACTGATGCGAAGA GGG -3 '(SEQ ID NO: 4)

Mouse-syndecan-2 sense sequence, 5'GGAGAAACAUUCAGACAAU-3 '(SEQ ID NO: 5)

Mouse-syndecan-2 antisense sequence, 5′AUUGUCUGAAUGUUUCUCC-3 ′ (SEQ ID NO: 6) boldface comprises a syndecan-2 sequence; Italics indicate hairpin loops. Scrambled small interfering RNA (siGENOME Non-Targeting siRNA # 2) was obtained from Dharmacon, Inc. (Chicago, IL).

Example 4 Analysis of Metastasis

Cells were detached from the culture dish for 5 minutes with PBS with 0.02% EDTA, centrifuged and resuspended with 200 μl of PBS. For experimental metastasis, cells (1 × 10 ⁵ / mouse) were injected into the tail vein of 5 week old female C57BL / 6 mice. Mice were sacrificed 13 days after injection and metastatic nodules were observed on the surface of the lungs.

Example 5: RNA Extraction and Reverse Transcription PCR

Total RNA extracted from the cultured cells was used as a template for reverse transcriptase reaction. cDNA was amplified using the following primers:

 Rat Syndecane-2 (Forward) 5'-ATGCGGGTACGAGCCACGTC -3 'and (Backward) (SEQ ID NO: 7)

 5'-CGGGAGCAGCACTAGTGAGG-3 '(SEQ ID NO: 8); Rat β-actin (forward) 5'-TGGAATCCTGTGGCATCCATGAAA-3 '(SEQ ID NO: 9) and (backward) 5'-TAAAACGCA GCTCAGTAACAGTCCG-3' (SEQ ID NO: 10). After initial denaturation at 94 ° C. for 5 minutes, 30 cycles of annealing at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and expansion at 72 ° C. for 60 seconds were performed. The reaction products were analyzed on 1.5% agiros gel. The amplified DNA fragments were cloned and sequenced to identify the PCR products.

Example 6 Immunization and Immunoblotting

The culture was washed twice with PBS and the cells were treated with a protease inhibitor cocktail (1 μg / ml aprotinin, 1 μg / ml antipain, 5 μg / ml leupeptin, 1 μg / ml pepstatin A, 20 μg / ml phenylmethylsulfonyl fluoride). ) Was disrupted in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 2 mM Na ₃ VO ₄ ). The lysate was clarified by centrifugation at 13,000 xg, 4 for 15 minutes, denatured with SDS sample buffer, heated and analyzed by SDS-PAGE. For immunoprecipitation, each sample containing 200 to 1,000 μg total protein was incubated with appropriate antibody for 2 hours at 4 ° C. and incubated with protein G-Sepharose beads for 1 hour. Immune complexes were collected by centrifugation. The proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and probed with appropriate antibodies. The signals were detected by enhanced chemiluminescence (Amersham Life Science).

Example 7: Slot Blotting

The culture was washed twice with PBS and the cells were washed with RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 2 mM Na ₃₎ containing a Protease Inhibitor Cocktail. VO ₄ ). The lysate was applied to a PVDF membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ) under mild vacuum in an immunoblot device (Slot blot, Bio-Rad). The membrane was then blocked by incubating for 1 hour at room temperature in TBS-T supplemented with 5% skim milk powder and then incubated at 4 with anti-sdc2 mAb. After washing, the membranes were incubated with species-specific HRP-conjugated secondary antibodies (Amersham Bioscience) and detection was performed using an ECL system (Ab Frontier, Korea).

Example 8: Flow Cytometry

Melanoma cells cultured in 60-mm diameter tissue culture dishes were washed with PBS, released by 1 mM EDTA, and then PBS with 5% FBS was added. After pelleting, cells were resuspended in PBS and counted. Cells (1 × 10 ⁶ / ml) were incubated with anti-synthecan-2 antibody for 1 hour at 4 ° C. in PBS with 10% FBS. After washing three times with PBS with 0.05% Tween-20, cells were incubated with FITC-conjugated anti-mouse antibody for 30 minutes in PBS with 10% FBS. Syndecane-2 expression was analyzed by flow cytometry.

Example 9 Movement Analysis

Gelatin (10 μg / ml) was added to each well of a Transewell plate (Costar; 8-μm aperture) and the membranes were dried at 25 ° C. for 1 hour. The Transwell plates were assembled in 24-well plates, and the bottom chamber was filled with FGF-2 (100 ng / ml) in fresh medium. Cells (5 × 10 ⁴ ) were added to each top chamber and the plates were incubated at 37 ° C., 5% CO ₂ for 12 hours. Cells that migrated to the bottom surface of the filter were stained and counted 0.6% hematoxylin and 0.5% eosin. For in vitro invasive analysis, 24-well Transwell plates (Costar; 8-μm aperture) were coated with gelatin (10 μg / ml) at the bottom of the membrane and Matrigel (30 μg / μl) at the top.

Example 10 Biopsy

Two milliliter punch biopsies were obtained from patients who provided their consent in accordance with the policies of the Catholic Medical Institutions Review Board. The punch biopsies were obtained from malignant lesions and normal epidermis of outpatients. Biopsies were immediately frozen in liquid nitrogen and kept at −70 ° C. until used for later analysis.

Example 11: Immunohistochemical Analysis

Five sets of biopsies obtained from the patient were fixed in 10% neutralized buffer formalin for 24 hours, placed in paraffin, sectioned and paraffin removed for staining. The sections were stained with hematoxylin and eosin. For immunostaining, the sections were washed and endogenous peroxidase activity was blocked in PBS with 3% hydrogen peroxide and 10% methanol, followed by 0.2% Triton X-100 (PBS-T) and 3% bovine serum albumin ( BSA) was preincubated for 2 hours in PBS. The sections were then incubated for 16 hours in PBS-T containing anti-SDC2 antibody (1: 1000) or anti-melanosome HBM-45 antibody (1:50). After washing with PBS, they were washed and incubated for 60 minutes at room temperature with biotinylated secondary antibody (1: 1,000, Dako, Denmark) in PBS-T with 3% bovine serum albumin (BSA). The sections were then incubated with peroxidase labeled streptavidin (Dako, Denmark) for 60 minutes and then reacted with diaminobenzidine (0.5 mg / ml) and hydrogen peroxide. After washing, the sections were fixed, dehydrated and covered with coverslips. All images were taken with Carl Zeiss Axioskop2.

Example 12 Construction of Transcript Syndecane-2 Reporter Plasmid

The 3 kb long syndecane-2 promoter gene was amplified by PCR using the following mouse syndecane-2 primers: (forward) 5 '

GGTACCATCTCATCAACACCAA-3 (SEQ ID NO: 11) and (BACKWARD) 5'AGGGATATGAGGCCATGGTAAT-3 '(SEQ ID NO: 12) After initial denaturation at 94 ° C for 5 minutes, annealing for 30 seconds at 30 cycles of 94 ° C, 30 seconds at 55 ° C, and 60 seconds of expansion was performed at 72 ° C. The syndecan-2 promoter gene was ligated with pGL3-basic vector at the Kpn / Xho cloning site.

Example 13: Luciferase Assay

B16 melanoma cells were plated in 24-well plates for 24 hours prior to transfection with 0.1 μg of pCMV / b-GAL as a control to standardize or with 0.2 μg of pGL3-basic vector. 24 hours after transfection, cells were washed with PBS and disrupted with Reporter Lysis Buffer (Promega). Aqueous extracts were pooled to determine luciferase and β-galactosidase activity according to the manufacturer's instructions. All transfections were repeated at least three times using different plasmid formulations.

Example 14 Wound-healing Analysis

Melanoma cells were plated on 60-mm tissue culture plates and cultured overnight. One streak was made in the middle of the monolayer using a sterile pipette tip and then the cells were washed twice with PBS and incubated in fresh medium. Photos of the wound distance were taken at various time intervals.

Example 15: Measurement of melanin levels

Melanin amounts were measured as reported by Kazuomi et al (Sato, K., Takahashi, H., Iraha, R., and Toriyama, M. (2008) Biol Pharm Bull 31 (1), 33-37). . In summary, cells were detached by incubating with trypsin / EDTA for 48 hours with 1 mM a-MSH or 200 mM Kojic acid in DMEM containing 10% heat inactivated FBS. After pelleting, cells were resuspended in DMEM for cell counting or lysed in boiling 2 M NaOH for 20 minutes. After cooling to room temperature, the absorbance of the solution was measured at 405 nm to determine the amount of melanin.

Example 16: Cell Proliferation Assay

Cell proliferation was measured by colorimetric analysis using MTT. MTT analysis was performed as described in (Park, H., Kim, Y., Lim, Y., Han, I., and Oh, ES (2002) J Biol Chem 277 (33), 29730-29736).

The result of the above Example is as follows.

To investigate the role of syndecane-2 in the development of human cutaneous melanoma, the expression of syndecane-2 was compared to tissues obtained from normal and malignant skin cancers. Immunohistochemical analyzes showed the absence of syndecane-2 expression in normal skin (FIG. 2A, b ). On the other hand, we found an increase in syndecane-2 expression in the dermal nevus (FIGS. 2A, f, g ). Interestingly, syndecan-2 expression was significantly increased in malignant melanoma positively stained with HMB-45, a well-known marker of melanoma in adjacent slides (FIGS. 2A, k, l ) (FIGS. 2A, d, h, i). , m, n ), whereas pre-immune serum was not stained except melanin granules in malignant melanoma (FIGS. 2A, c ). Consistent with this observation, increased cell surface expression of syndecane-2 was observed in human melanoma cell lines (FIG. 2B). These data suggest that syndecane-2 expression is increased in malignant melanoma and that syndecane-2 expression promotes melanin synthesis.

In order to directly assess the role of syndecane-2 in the regulation of oncogenic activity, we investigated the effect of syndecane-2 expression on melanoma cell migration. Human melanoma A375 cells were transfected with syndecan-2 cDNA (S2W) and transwell migration assays were performed (FIG. 3A). After transfection with S2W, cell migration was significantly higher in A375 cells than vector-transfected cells (ie vec). On the other hand, cell migration was reduced when syndecan-2 expression was inhibited by small inhibitory RNAs (siRNAs) (FIG. 3B). As with human melanoma cells, increased cell surface expression of syndecane-2 was also observed in mouse melanoma cell lines (FIG. 4A). Syndecan-2 overexpression increased migration of mouse melanoma B16 cells based on transwell migration (FIG. 4A) and wound-healing analysis (FIG. 4B), whereas syndecan-2 expression was induced by small inhibitory RNAs (siRNAs). Cell inhibition was reduced when inhibited (FIG. 4C). Knockdown of syndecane-2 expression by siRNA reduced the metastatic capacity of B16F10 melanoma cells in mice (FIG. 4D). These findings strongly indicate that dedecane-2 regulates cell migration in melanoma cells.

Focal adhesion kinase (FAK) is known to play an important role in cancer cell migration (Owens, LV, Xu, L., Dent, GA, Yang, X., Sturge, GC, Craven, RJ, and Cance, WG. (1996) Ann Surg Oncol 3 (1), 100-105; Owens, LV, Xu, L., Craven, RJ, Dent, GA, Weiner, TM, Kornberg, L., Liu, ET, and Cance, WG. (1995) ) Cancer Res 55 (13), 2752-2755). We found that whole cell tyrosine phosphorylation increased in S2W-overexpressing B16 melanoma cells (FIG. 5A). Increased tyrosine phosphorylation mediated by FAK often occurs in response to promoting cell migration (Park, H., Han, I., Kwon, HJ, and Oh, ES (2005) Cancer Res 65 (21), 9899-9905 ). Tyrosine phosphorylation (ie p120, p80, p60, and p50) of the four types of polypeptides was higher in cells transfected with S2W than in cells transfected with control vectors. In addition, S2W expression can be attributed to autophosphorylation of tyrosine 397 (ie, Y397), phosphorylation of phosphorylation of FAK tyrosine 861 (ie, Y861 is known to regulate cellular transformation) (FIG. 5B), and paxillin, well known as a substrate of FAK. Increased tyrosine phosphorylation (FIG. 5C). These data indicate that syndecane-2-induced migration activity is associated with activation of FAK-mediated signaling.

To further investigate the regulation of syndecan-2 expression, we cloned the syndecan-2 promoter sequence (ie, directly upstream of the 3 kb region of the gene) into a luciferase expressing plasmid containing the firefly luciferase reporter gene. (FIG. 6A). Plasmids containing an empty vector or syndecan-2 promoter were transiently transfected into B16 mouse melanoma cells and luciferase activity against FGF-2 was measured. We observed cell surface expression and increased luciferase activity in response to FGF-2 treatment (FIG. 6B). Migration of melanoma cells was also increased by FGF-2 treatment (FIG. 6D). These results indicate that syndecane-2 expression correlates with FGF-2-increased cell migration.

Bovine pituitary extract (BPE) is commonly used as a mitogenic supplement in growth media for human melanocytes (Donatien, P., Surleve-Bazeille, JE, Thody, AJ, and Ta ◎ b, A. ( 1993) Arch Dermatol Res 285 (7), 385-392). In contrast to FGF-2 treatment, BPE treatment reduced syndecan-2 luciferase activity, cell surface expression of syndecan-2 (FIG. 6C) and melanoma cell migration (FIG. 6D), which resulted in syndecan-2 expression. It was correlated with this melanoma cell migration. Since BPE contains α-MSH, we investigated whether α-MSH inhibits inhibits syndecane-2 expression in melanoma cells. As expected, α-MSH decreased cell surface expression of syndecane-2 (FIGS. 7A-C). Consistent with decreased syndecane-2 expression, α-MSH reduced the migration and invasion of melanoma cells (FIG. 7D & 7E), whereas cell proliferation was not significantly affected by α-MSH treatment for 24 hours. (FIG. 7F). In addition, syndecane-2 overexpression restored the inhibition of migration induced by a-MSH treatment (FIG. 8). Collectively, these results support that a-MSH inhibits melanoma cell migration through reduced expression of syndecane-2 and that syndecane-2 expression plays an important role in melanoma cell migration.

Example 17 Mobility in Melanocytes

Cell Culture and Transfection

Human primary melanocyte HEM cells were MGM-4 media (LonZa), mouse malignant melanoma cell line B16F10 was DMEM (Life Technologies) media with 10% FBS and 50 μg / ml gentamicin, and another mouse malignant black The species cell line, B16G4F, was cultured in a humidified incubator at 37 ° C and 5% CO ₂ in DMEM (Life Technologies) medium supplemented with 200 nM L-glutamine, 10% FBS and 50 µg / ml gentamicin. Transient transfection was performed with LipofecATMINE 2000 (Invitrogen) according to the manufacturer's instructions provided.

Small interfering RNA production

Oligonucleotides designed to target mouse syndecane-2 are sense sequences, 5-GGAGAAACAUUCAGACAAU-3 (SEQ ID NO: 5) antisense sequences, 5-AUUGUCUGAAUGUUUCUCC-3 (SEQ ID NO: 6).

Tissue

2mm general skin tissue and malignant melanoma lesion skin tissue were received from patients who visited Catholic University Gangnam St. Mary's Hospital and agreed with the Catholic Medical Research Association's policy. Biological tissues were immediately frozen in liquid nitrogen and stored at 70 ° C.

Tissue immunostaining

Five sets of biological tissues received from patients were fixed in 10% neutralized buffered formalin for 24 hours, placed in paraffin, dissected and paraffin removed for staining. The sections were washed and the endogenous peroxidase activity was inhibited with a solution of 3% hydrogen peroxide and 10% methanol in PBS, 0.2% Triton X-100 (PBS-T) and 3% bovine serum albumin ( BSA) was incubated for 2 hours with PBS added. Sections were then incubated for 16 hours with PBS-T containing anti-synthecan-2 (1: 1000) antibody or anti-melanosome HMB-45 antibody (1:50). After washing with PBS, the biotinylated second antibody (1: 1,000, Dako) in PBS-T to which 3% BSA was added was reacted at room temperature for 60 minutes. The tissue sections were rinsed properly and then reacted with peroxidase conjugated streptavidin (Dako) for 60 minutes, followed by diaminobenzidine (0.5 ㎍ / ml) and hydrogen peroxide. After another rinse, the tissue sections were fixed on slides, dehydrated, covered with coverslip, and tissue photographs were taken with Carl Zeiss Axioskop2.

Migration assay

Gelatin (10 μg / ml) was coated on the memebrane of each well of Transewell plate (Costar; 8-pore size) and dried at 25 ° C. for 1 hour. The Transwell plate consisted of 24 wells and the lower chamber was filled with fresh medium added with FGF-2 (100 ng / ml). 1 × 10 ⁵ cells were placed in each stomach chamber and plates were incubated for 24 hours at 37 ° C., 5% CO ₂ conditions. Cells migrated to the lower surface of the filter in the upper chamber were stained with 0.6% hematoxylin and 0.5% eosin and counted. For in vitro invasion analysis, the lower side of a 24-well transwell plate (Costar; 8-pore size) transwell membrane is coated with gelatin (10 μg / ml) and the upper side with Matrigel (30 μg / μl) After that it proceeded.

Flow cytometry

Cells cultured in 60 mm culture plates were rinsed with PBS and dropped into PBS containing 1 mM EDTA and 5% FBS. After pelleting the cells were re-dissolved in PBS and counted. 1 X 10 ⁶ / ml cells were reacted in PBS containing 10% FBS with anti-synthecan-2 antibody for 12 hours at 4 ° C. After rinsing three times with PBS containing 0.05% Tween-20, the cells were reacted with FITC conjugated anti-mouse antibody in PBS containing 10% FBS for 30 minutes at room temperature. Syndecane-2 expression was analyzed by flow cytometry.

The production of melanin pigment is promoted by α-MSH in skin, hair and eyes (Price, ER, Horstmann, MA, Wells, AG, Weilbaecher, KN, Takemoto, CM, Landis, MW, and Fisher, DE (1998) J Biol Chem 273 (49), 33042-33047) . α-MSH promotes upregulation of intracellular cAMP and subsequent activation of several pigmentation enzymes (eg tyrosinase catalyzing the rate step in melanin synthesis) (Passeron, T., Valencia, JC). , Bertolotto, C., Hoashi, T., Le Pape, E., Takahashi, K., Ballotti, R., and Hearing, VJ (2007) Proc Natl Acad Sci USA 104 (35), 13984-13989). We therefore investigated whether melanin synthesis plays an important role in the syndecan-2-mediated cell migration of melanoma cells. α-MSH increased the melanin amount of melanoma cells (ie approximately 121%), while melanin production was reduced by the tyrosinase inhibitor kojic acid (ie approximately 76%) (FIG. 9A). On the other hand, syndecane-2 expression was decreased by a-MSH treatment but not changed by kojic acid (FIGS. 9B & 9C). Thus, α-MSH-mediated reduction in cell migration is consistent with decreased syndecane-2 expression, but inhibition of tyrosinase activity did not affect melanoma cell migration (FIG. 9D). Α-MSH thus indicates that it inhibits syndecane-2-mediated cell migration independent of melanin biosynthetic pathway.

Example 18 Comparison of Melanin Synthesis by Syndecan-2 Expression :

In the case of comparing melanin synthesis after knocking down syndecane-2 by treating mouse syndecane-2 siRNA (SEQ ID NO: 5 and SEQ ID NO: 6) to B16F10 malignant melanoma with high expression of syndecane-2, Melanin synthesis was decreased in cells with reduced decan-2 expression (data omitted). It was observed that the synthesis of melanin increased when artificially overexpressing mouse-syndecan-2 cDNA again in syndecan-2 known-down cells (FIG. 12).

Example 19 Observation of Melanin Synthesis Using Tissue Immunostaining

Normal and cancerous skin tissues were stained with synthecan-2 antibody and melanosome marker HBM-45, respectively, to investigate the correlation between synthecan-2 expression and melanin synthesis. As a result, it was observed that melanin granules were increased in melanocytes and melanoma with high syndcan-2 expression (FIG. 13).

 Overexpression of syndecane-2 in human primary melanocytes resulted in an increase in the expression of syndecane-2 on the cell surface compared to the control group, and increased cell mobility and invasiveness.

Meanwhile, in FIG. 12A, B16F10 and B16G4F cells are settled in a centrifuge, while B16F4 cells are black pellets while B16G4F cells are white pellets. This shows that B16F10 cells synthesize more melanin than B16G4F cells. In addition, it can be seen that the expression level of syndecane-2 on the cell surface is very high in B16F10 cells compared to B16G4F cells. B of 12 inhibits the expression of endogenous syndecane-2 by B16F10 cells using small interfering RNA, transfects the empty vector and synthecan-2 cDNA, and then sinks each cell using a centrifuge. The pellets of the -2 overexpressing cells are blacker than the vector cells, indicating more melanin synthesis.

FIG. 13 shows immunohistostaining in order to determine the degree of syndecan-2 expression and melanosome formation in biological tissues. The expression of syndecane-2 is very high, and melanosome formation is also active.

Therefore, the above results can be seen that the increase in syndecan-2 expression is associated with increased melanin synthesis.

In the present invention, the melanin synthesis amount measurement method after collecting the cells in the tube, treated with 1N NaOH / 10% DMSO and reacted for 2 hours at 80 ℃, solublization, centrifugation (12,000g, 10 minutes) The pellet was left, and the supernatant was collected and transferred to a new tube. The melanin content of the supernatant prepared at 470 nm was measured after standard curves were prepared at a concentration of 0-20 ug / ml using synthetic or purified melanin.

1 is a characterization of monoclonal antibodies against syndecane-2. Schematic description of A, GST-syndecan-2 and -4 core proteins. Extracellular domains are indicated by white boxes (EC), transmembrane domains by black boxes (TM) and plasma domains by gray boxes (CT). Syndecane-2 and -4 are labeled with amino acid numbers indicating the site of the deletion mutant. B, GST-syndecan-2 core proteins were expressed in E. coli. Purified recombinant proteins were isolated on 10% SDS-PAGE and immunoblotted with the indicated Sindecan-2 antibody. C, HCT116 colon cancer cells were incubated with the indicated syndecane-2 antibody and cell surface expression levels of syndecane-2 were analyzed by flow cytometry (top panel). IgG was used as a control. Total cell lysate obtained from HCT116 was analyzed by slot blot with anti-synthecan-2 antibody (mAb # 197, bottom panel). D, HT-29 colon cancer cells and whole cell lysate obtained from HT-29 stably expressing syndecane-2 (HT-29-sdc2) were analyzed by slot blotting with the indicated syndecane-2 antibody (Top, Left panel). Cells were incubated with anti-synthecan-2 antibody (mAb # 197), stained with Texas Red-conjugated secondary antibody and photographed under confocal microscopy (top, right panel). Cells were incubated with the indicated syndecane-2 antibody and cell surface expression levels of syndecane-2 were analyzed by flow cytometry. IgG was used as a control (bottom panel). Murine syndecane-2 expressed in HT-29 cells is recognized by both mAbs # 197 and # 302, while endogenous human syndecane-2 in HCT116 cells is specifically recognized by mAb # 197.

2 shows that syndecane-2 is highly expressed in human skin melanoma cells. A, paraffin-filled tissue sections were stained with hematoxylin and eosin (H & E, a), hematoxylin (e and j), or immunostained with anti-synthecan-2 antibody (SDC2 mAb # 197, b, f, k). c is a negative control for immunostaining with pre-immune serum. Immune response was visualized with 3,3'-Diaminobenzidine (DAB) enhanced by nickel. g and i are larger magnifications of f and k, respectively. Immunostaining with anti-HMB-45 antibody was detected by the same method (HBM-45, d, h, m). i and n are larger magnifications of h and m, respectively. Arrows indicate melanocytes with immunoreactivity of syndecan-2 and HMB-45, respectively. The figures represent a representative of one of five experiments. B, cells obtained from human melanoma cell line were incubated with anti-synthecan-2 antibody and protein expression levels were analyzed by flow cytometry. IgG was used as a control.

3 shows that syndecane-2 regulates human melanoma migration. A375 human melanoma cells were transfected with 2 μg sdc2 cDNA (A) or si-syndecane-2 plasmid (B). Total cell lysate was analyzed by slot blotting with anti-syndecane-2 antibody (top panel). Cells were incubated with anti-synthecan-2 antibody and cell surface expression levels of syndecane-2 were analyzed by flow cytometry. IgG was used as a control (middle panel). Transwell migration assays were performed with directed A375 cells using FGF-2 as chemoattractant in the lower chamber. The cells (5 × 10 ⁴ ) are allowed to migrate for 12 h in a 10 μg / ml gelatin coated Transwell plate. After fixation and staining with 0.6% hematoxylin and 0.5% eosin, the number of migrated cells was counted. Column, average of at least 3 independent experiments (bottom panel). _* p <0.01, _** p <0.05 versus control. For invasive assays, cells were loaded into the top section of Matrigel (30 μg / ml) coated plates and incubated for 12 hours.

4 shows that Sindecane-2 regulates mouse melanoma cell migration. A , B16 cells were transfected with syndecan-2 cDNA. Total RNA was extracted and expression of syndecane-2 was analyzed by reverse transcription-PCR. GAPDH mRNA was used as a loading control (top panel). Cells were incubated with anti-synthecan-2 antibody and cell surface expression levels of syndecane-2 were analyzed by flow cytometry. IgG was used as a control (middle panel). As described in FIG. 3, the Transwell migration assay was performed with the indicated B16 cells using FGF-2 as chemoattractant in the lower chamber (bottom panel). B, B16 cells were transfected with syndecan-2 cDNA and after 24 hours, wound-healing analysis was performed. One screak was made in the center of the monolayer using a sterile pipette tip and a photograph of the wound distance was taken at each time point. The cells (5 × 10 ⁴ ) are allowed to migrate to the Transwell plate as described in FIG. 3. Column, average of at least 3 independent experiments. _* p <0.01 versus control. D, B16F10 cells were transfected with syndecane-2 si-RNA. Cells were injected intravenously into (1 × 10 ⁵ ) 5 week old c57BL / 6 mice. Mice were sacrificed 13 days after injection. Metastatic nodules on the surface of the lung were observed.

5 shows that Focal adhesion kinase is involved in the regulation of syndecane-2-mediated melanoma cell migration. A , empty vector (control) or B16 cells transfected with Cintecan-2 cDNA (sdc2) were disrupted with RIPA buffer and westernized with anti-phosphotyrosine (a-PY) antibody and anti-vinculin antibody after scraping. Analysis by blot. B , B16 cells were disrupted and site-specific FAK phosphorylation was analyzed with anti-FAK [PY ³⁹⁷ ] and anti-FAK [PY ⁸⁶¹ ] antibodies, stripped and rerobed with anti-FAK antibodies. C , B16 cell extracts were immunoprecipitated with an antibody against paxillin, and the phosphorylation status of the protein was determined by immunoblotting with an anti-phosphotyrosine (PY) antibody. The level of protein in each immunoprecipitator was determined by immunoblotting with anti-paxillin antibody.

6 shows that syndecane-2 expression correlates with melanoma cell migration. A , depiction of the Cindecan-2 promoter construct having the 5'-franking site of mouse syndecan-2 promoter sequences linked to the firefly luciferase reporter gene. B16 cells transfected with B , C , Cindecan-2 reporter plasmid were treated with BPE (0.4% v / v) or FGF-2 (300 ng / ml) for 24 hours and luciferase activity was measured. Column, average of at least 3 independent experiments (top panel). Cells were incubated with anti-synthecan-2 antibody and their protein expression levels analyzed by flow cytometry. IgG was used as a control (bottom panel). Cells treated with D, BPE or FGF-2 (5 × 10 ⁴ ) were allowed to migrate on Transwell plates as described in FIG. 3. Column, average of three independent experiments. _* p <0.01, _** p <0.05 versus control.

FIG. 7 shows that α-MSH inhibits the expression of syndecane-2 as it reduces cell migration. A , B16 cells were treated with α-MSH (1.0 μM) for 24 hours. Total RNA was extracted and expression of syndecane-2 was analyzed by reverse transcription-PCR. β-actin mRNA was used as loading control. B , B16 cells were treated with FGF-2 (300 ng / ml) or α-MSH (1.0 μM). After 24 hours, total cell lysate was analyzed by slot blotting. C , B16 cells treated with indicated amounts of α-MSH (1.0 μM) for 24 hours were incubated with anti-synthecan-2 antibody (mAb # 197) and the protein expression levels were analyzed by flow cytometry. IgG was used as a negative control D , B16 cells (5 × 10 ⁴ ) were pretreated with FGF-2 (300 ng / ml) or α-MSH (0.5 μM, 1.0 μM) for 24 hours and then those described in FIG. 3. Move on the transwell plate as well. Column, average of three independent experiments. E , B16 cells were pretreated with α-MSH (1.0 μM) for 24 hours. Transwell cell migration or invasion assay was performed as in FIG. 3. Column, average of three independent experiments. F , B16 cells were treated with FGF-2 (300 ng / ml) or α-MSH (1.0 μM), followed by MTT assay. _* p <0.01, _** p <0.05 versus control.

8 shows that α-MSH inhibits melanoma cell migration through reduced expression of syndecane-2. A , B16 cells were transfected with empty vector or syndecane-2 cDNA after treatment for 24 hours with α-MSH (1.0 μM). Syndecane-2 expression levels (A) and cell migration (B) were analyzed as described in FIG. 3. Column, average of three independent experiments.

9 shows that syndecane-2-mediated cell migration is independent of melanin synthesis. A , B16F10 cells were treated with α-MSH (1.0 μM) or kojic acid (200 μM) for 48 hours, and the melanin content of the cells was measured as in Example. Column, average of three independent experiments. B , B16F10 cells transfected with a Sindecan-2 reporter construct were treated with 1.0 μM of α-MSH or 200 μM of kojic acid for 48 hours. Syndecane-2 promoter activity was analyzed by luciferase assay. C , B16F10 cells were treated with α-MSH (1.0 μM) or kojic acid (200 μM) for 48 hours. Cell surface expression (top panel) and cell migration activity of syndecane-2 (bottom panel) were analyzed as in FIG. 3. _* p <0.05, _** p <0.01 versus control.

10 is a diagram illustrating the effect of controlling the cell mobility of natural products using an artificial skin model.

11 is a diagram showing that syndecane-2 increases cell mobility of melanocytes. Melanocytes were cultured in 60 mm culture plates and transfected with empty vector and syndecan-2 cDNA, respectively, for 48 hours, followed by flow cytometry, transwell cell migration and infiltration analysis. After the cells were reacted with the Sindecan-2 antibody, the expression level of the Sindecan-2 on the cell surface was analyzed by flow cytometry. IgG was used as a control (top panel). Transwell cell migration and infiltration assays were performed for 24 hours using 1 × 10 ⁵ cells as described in FIG. 3 (bottom panel).

12 is a diagram showing that syndecane-2 increases melanin synthesis in malignant melanoma cell lines. A , mouse malignant melanoma cell lines B16F10 and B16G4F were each incubated in a 60 mm culture plate, then allowed to settle by centrifuge and photographed pellets (top panel), and the cells were reacted with syndecan-2 antibody and then placed on the cell surface. The amount of syndecane-2 expression was analyzed using a flow cytometer. IgG was used as a control (bottom panel). B , B16F10 cells were co-transfected with the dedecane-2 si-RNA and the empty vector or the dedecane-2 cDNA, the cells were allowed to settle by centrifuge, and the pellets were photographed.

FIG. 13 is a diagram showing that syndecane-2 is highly expressed in malignant melanoma and melanin synthesis is also increased. Paraffin-filled tissue sections were immunostained with anti-synthecan-2 antibodies (SDC2, a, b ) and anti-HMB-45 antibodies ( c , d ). Arrows indicate where strong immune responses from syndecane-2 and HMB-45 are indicated. The pictures show only one representative photograph of five experiments.

<110> EHWA UNIVERSITY INDUSTRY COLLABORATION FOUNDATION <120> A composition for preventing or treating migratory skin cell or          melanin synthesis related diseases <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 201 <212> PRT <213> Homo sapiens <400> 1 Met Arg Arg Ala Trp Ile Leu Leu Thr Leu Gly Leu Val Ala Cys Val   1 5 10 15 Ser Ala Glu Ser Arg Ala Glu Leu Thr Ser Asp Lys Asp Met Tyr Leu              20 25 30 Asp Asn Ser Ser Ile Glu Glu Ala Ser Gly Val Tyr Pro Ile Asp Asp          35 40 45 Asp Asp Tyr Ala Ser Ala Ser Gly Ser Gly Ala Asp Glu Asp Val Glu      50 55 60 Ser Pro Glu Leu Thr Thr Ser Arg Pro Leu Pro Lys Ile Leu Leu Thr  65 70 75 80 Ser Ala Ala Pro Lys Val Glu Thr Thr Thr Leu Asn Ile Gln Asn Lys                  85 90 95 Ile Pro Ala Gln Thr Lys Ser Pro Glu Glu Thr Asp Lys Glu Lys Val             100 105 110 His Leu Ser Asp Ser Glu Arg Lys Met Asp Pro Ala Glu Glu Asp Thr         115 120 125 Asn Val Tyr Thr Glu Lys His Ser Asp Ser Leu Phe Lys Arg Thr Glu     130 135 140 Val Leu Ala Ala Val Ile Ala Gly Gly Val Ile Gly Phe Leu Phe Ala 145 150 155 160 Ile Phe Leu Ile Leu Leu Leu Val Tyr Arg Met Arg Lys Lys Asp Glu                 165 170 175 Gly Ser Tyr Asp Leu Gly Glu Arg Lys Pro Ser Ser Ala Ala Tyr Gln             180 185 190 Lys Ala Pro Thr Lys Glu Phe Tyr Ala         195 200 <210> 2 <211> 606 <212> DNA <213> Homo sapiens <400> 2 atgcggcgcg cgtggatcct gctcaccttg ggcttggtgg cctgcgtgtc ggcggagtcg 60 agagcagagc tgacatctga taaagacatg taccttgaca acagctccat tgaagaagct 120 tcaggagtgt atcctattga tgacgatgac tacgcttctg cgtctggctc gggagctgat 180 gaggatgtag agagtccaga gctgacaaca tctcgaccac ttccaaagat actgttgact 240 agtgctgctc caaaagtgga aaccacgacg ctgaatatac agaacaagat acctgctcag 300 acaaagtcac ctgaagaaac tgataaagag aaagttcacc tctctgactc agaaaggaaa 360 atggacccag ccgaagagga tacaaatgtg tatactgaga aacactcaga cagtctgttt 420 aaacggacag aagtcctagc agctgtcatt gctggtggag ttattggctt tctctttgca 480 atttttctta tcctgctgtt ggtgtatcgc atgagaaaga aggatgaagg aagctatgac 540 cttggagaac gcaaaccatc cagtgctgct tatcagaagg cacctactaa ggagttttat 600 gcgtaa 606 <210> 3 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gatcccctga cgatgactac gcttctttca agagaactgc tactgatgcg aagatttttg 60 gaaa 64 <210> 4 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agcttttcca aaaatgacga tgactacgct tcttctcttg aaactgctac tgatgcgaag 60 aggg 64 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 5 ggagaaacau ucagacaau 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA <400> 6 auugucugaa uguuucucc 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 atgcgggtac gagccacgtc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cgggagcagc actagtgagg 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tggaatcctg tggcatccat gaaa 24 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 taaaacgcag ctcagtaaca gtccg 25 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggtaccatct catcaacacc aa 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agggatatga ggccatggta at 22  

Claims (9)

A composition for the treatment or prevention of mobile skin diseases comprising syndecane-2 consisting of SEQ ID NO: 1 as an active ingredient. The method of claim 1, wherein the mobile skin disease is a composition for the treatment or prevention of mobile skin diseases, characterized in that at least one disease selected from the group consisting of melanoma, skin pigmentation, skin pigmentation and vitiligo. Syndecane-2 consisting of SEQ ID NO: 2 A composition for treating or preventing mobile skin diseases comprising a gene as an active ingredient. The mobile skin disease of claim 3, wherein the mobile skin disease is one or more diseases selected from the group consisting of melanoma, skin pigmentation, skin pigmentation, and vitiligo. Therapeutic or prophylactic composition. A composition for the treatment or prevention of mobile skin diseases, comprising as an active ingredient a gene for one or more syndecane-2 selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 6. The mobile skin disease of claim 5, wherein the mobile skin disease is at least one disease selected from the group consisting of melanoma, skin pigmentation, skin pigmentation, and vitiligo. Therapeutic or prophylactic composition. Skin whitening composition comprising a syndecane-2 consisting of SEQ ID NO: 1 as an active ingredient. A composition for skin whitening comprising a syndecane-2 gene consisting of SEQ ID NO: 2 as an active ingredient. A composition for skin whitening comprising as an active ingredient a gene for at least one syndecane-2 selected from the group consisting of SEQ ID NOs: 3 to 6.

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